The fast polarization modulation based dualfocus fluorescence correlation spectroscopy Martin Štefl,1,4 Aleš Benda,1,3,4,* Ingo Gregor,2 and Martin Hof1 1

J. Heyrovsky Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Dolejškova 3, Prague 182 23, Czech Republic 2 Georg-August-Universität Göttingen, 3rd Institute of Physics, Friedrich-Hund-Platz 1, D-37077 Göttingen, Germany 3 Centre for Vascular Research and Australian Centre for NanoMedicine, University of New South Wales, Sydney, NSW 2052, Australia 4 Authors contributed equally to this work * [email protected]

Abstract: We introduce two new alternative experimental realizations of dual focus fluorescence correlation spectroscopy (2fFCS), a method which allows for obtaining absolute diffusion coefficient of fast moving fluorescing molecules at nanomolar concentrations, based on fast polarization modulation of the excitation beam by a resonant electro-optical modulator. The first approach rotates every second linearly polarized laser pulse by 90 degrees to obtain independent intensity readout for both foci, similar to original design. The second approach combines polarization modulation of cw laser and fluorescence lifetime correlation spectroscopy (FLCS) like analysis to obtain clean correlation curves for both overlapping foci. We tested our new approaches with different lasers and samples, revealed a need for intensity cross-talk corrections by comparing the methods with each other and discussed experimental artifacts stemming from improper polarization alignment and detector afterpulsing. The advantages of our solutions are that the polarization rotation approach requires just one pulsed laser for each wavelength, that the polarization modulation approach even mitigates the need of pulsed lasers by using standard cw lasers and that it allows the DIC prism to be placed at an arbitrary angle. As a consequence the presented experimental solutions for 2fFCS can be more easily implemented into commercial laser scanning microscopes. © 2014 Optical Society of America OCIS codes: (170.6280) Spectroscopy, fluorescence and luminescence; (180.1790) Confocal microscopy; (300.2530) Fluorescence, laser-induced.

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#200468 - $15.00 USD (C) 2014 OSA

Received 30 Oct 2013; revised 16 Dec 2013; accepted 19 Dec 2013; published 8 Jan 2014 13 January 2014 | Vol. 22, No. 1 | DOI:10.1364/OE.22.000885 | OPTICS EXPRESS 885

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1. Introduction Determination of diffusion coefficients in live cells or in model systems is usually achieved by fluorescence based methods like Fluorescence Recovery After Photobleaching (FRAP) [1], Fluorescence Correlation Spectroscopy (FCS) [2] [3] or fluorescence Single Particle Tracking (SPT) [4]. These methods are highly complementary, each exploring different concentration, spatial and also time ranges. A strong feature of FCS is its concentration range mostly suiting molecules abundance under cellular conditions (micromolar to nanomolar concentrations) and its coverage of long time span from nanoseconds to seconds. There exist multitudes of different realizations of FCS experiments, from simple fixed single focus single color FCS, through fixed multi-color [5] and lifetime FCS [6] or scanning FCS variants [7] to image correlation approaches like Raster Image Correlation Spectroscopy (RICS) [8]. Each variant suits different sample and experimental conditions. For studies requiring precise artifact free determination of diffusion coefficients (D) of small and middle size molecules freely diffusing in solvent (D = 10−10 – 10−13 m2s−1) or laterally diffusing within a lipid membrane (D = 10−12 – 10−13 m2s−1), dual-focus FCS is certainly the method of choice. Dual-focus Fluorescence Correlation Spectroscopy (2fFCS) has become a very robust tool for determining absolute values of diffusion coefficients of fluorescent species in dilute solutions [9]. Its robustness comes from the introduction of an external ruler – a precisely known distance between two foci, which is insensitive to various aberrations coming from both the microscope and the measured sample. It has been shown that 2fFCS allows for precise determination of diffusion coefficients under strongly varying experimental conditions (temperature, refractive index of media, focal plane position alterations), where standard approaches fail [9].

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Received 30 Oct 2013; revised 16 Dec 2013; accepted 19 Dec 2013; published 8 Jan 2014 13 January 2014 | Vol. 22, No. 1 | DOI:10.1364/OE.22.000885 | OPTICS EXPRESS 886

The key aspect of 2fFCS is to have two overlapping foci, but still being able to distinguish fluorescence emitted from each of them. The standard approach [9] uses two pulsed interleaved lasers with crossed polarizations, which after passing a differential interference contrast (DIC) prism create two spatially overlapping, but time separated foci. The first attempt to simplify the hardware requirements for 2fFCS used an excitation beam polarization modulation at 100 kHz frequency, allowing for using cw lasers without the need of TimeCorrelated Single Photon Counting (TCSPC) electronics [10]. The rather low frequency polarization modulation does not allow to separate intensity from both foci and instead of fitting clean auto- and cross- correlation functions for both foci a single auto-correlation curve is fitted by a more complex model accounting for the modulated mixing of auto- and crosscorrelations. In this contribution we demonstrate two new approaches, based on utilization of fast 20 MHz electro-optical modulator (EOM), to experimentally perform 2fFCS measurement. The first polarization rotation approach uses EOM to rotate polarization of every second laser pulse by 90 degrees, allowing 2fFCS setup with only one pulsed laser of each color. The second polarization modulation approach combines EOM polarization modulation of a cw laser with filtered fluorescence correlation analysis (filtered-FCS) [11], which can be considered as a generalized version of fluorescence lifetime correlation spectroscopy (FLCS) [12,13]. The polarization modulation of cw excitation beam is transformed by a DIC prism into two different excitation patterns (harmonic waves mutually shifted by a halfwave) for each focus. Knowing these patterns filtered-FCS analysis allows for auto- and crosscorrelation of fluorescence from both foci, giving the same type of data as the pulsed version of 2fFCS. We show that these new approaches give correct diffusion coefficients both for 3-D diffusion in solution and for 2-D membrane lateral diffusion. The presented approaches have the potential of widespread utilization in commercial laser scanning confocal microscopes, as they require only slight modification of the excitation path, an insertion of a compact EOM element, and require just one pulsed laser instead of two for polarization rotation approach, or even no pulsed laser at all, using only standard cw lasers, for polarization modulation approach. 2. Materials and methods 2.1 Materials 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-(1’rac-glycerol) (DOPG), cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamineN-(cap biotinyl) (biotin-DPPE) were purchased from Avanti Polar Lipids (Alabaster, AL). N(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)-1,2-dihexadecanoylsn-glycero-3-phosphoethanolamine, triethylammonium salt (BODIPY® FL DHPE), Alexa Fluor® 488 NHS-ester, 5-carboxytetramethylrhodamine (5-TAMRA) and TetraSpeckTM microspheres (200 nm) were obtained from Invitrogen (Carlsbad, CA). Atto 425 with free carboxy group was obtained from ATTO-TEC (Siegen, Germany). Perylene, 5-carboxyfluorescein, streptavidin, biotin-labeled bovine serum albumin (BSA-biotin), 4-(2Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), sodium chloride (NaCl), calcium chloride (CaCl2), ethylenediaminetetraacetic acid (EDTA), glucose and sucrose were purchased from Sigma (St. Louis, MO). 2.2 Supported phospholipid bilayer (SPB) and giant unilamellar vesicle (GUV) preparation SPBs: First, we prepared the mixture of appropriate lipids (final lipid concentration 2 mM), which contained the fluorescence dye in the lipid to dye ratio 200 000:1. The organic solvent was evaporated under the stream of nitrogen and thin lipid film was further kept for additional 2 h under the vacuum. The dried lipid film was subsequently hydrated with 10 mM HEPES buffer (150 mM NaCl, 1 mM EDTA, pH = 7.4) and the solution was extensively vortexed for

#200468 - $15.00 USD (C) 2014 OSA

Received 30 Oct 2013; revised 16 Dec 2013; accepted 19 Dec 2013; published 8 Jan 2014 13 January 2014 | Vol. 22, No. 1 | DOI:10.1364/OE.22.000885 | OPTICS EXPRESS 887

at least 2 min until multilamellar vesicles were formed. Next, the cloudy solution was sonicated for 20 min, yielding a solution of small unilamellar vesicles (SUVs). The SUVs were 10x diluted in the buffer containing Ca2+ ions (10 mM HEPES, 150 mM NaCl, 1 mM CaCl2, pH = 7.4), moved to the cuvette with glass surface and incubated for 60 min. The redundant vesicles were flushed and the cuvette with SPBs was placed directly on the microscope objective and measured. The SPBs used in the measurements were composed of 75 mol% of DOPC and 25 mol% of cholesterol. The lateral diffusion of perylene (excitation 405 nm) and BODIPY® FL DHPE (excitation 470 nm) was monitored. GUVs: GUVs were prepared by a gentle hydration method as described before [14]. 1 mL of desired lipids dissolved in chloroform and containing 1 mg of lipids was dried and placed under the vacuum for additional 2 h. Thin lipid film was hydrated with 3 mL of buffer (10 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 0.1 M sucrose, pH = 7). The tube was then sealed, heated up to 50 °C, kept overnight at this temperature, and slowly cooled down. White cloudy solution was gently vortexed before further use. All the prepared lipid mixtures contained 5 mol% of DOPG, negatively charged lipid necessary for the given preparation technique and 4 mol% of biotin-DPPE, which is needed for immobilization of GUVs to BSAbiotin/streptavidin coated glass surface. The BSA-biotin/streptavidin coated Lab-Tek chamber (NUNC A/S, Denmark) was filled with 380 μL of a buffer solution (10 mM HEPES, 150 mM NaCl, 1 mM CaCl2, 0.1 M glucose, pH = 7), 20 μL of the solution containing GUVs were added and after 30 min of incubation the measurements were performed. The GUVs were composed of 66 mol% of DOPC, 25 mol% of cholesterol, 5 mol% of negatively charged lipid DOPG and 4 mol% of biotin-DPPE. The lateral diffusion of BODIPY® FL DHPE (excitation 470 nm) was followed. 2.3 Experimental setup All experiments were carried out on a home-built confocal microscope setup (Fig. 1) based on an inverted microscope body IX71 (Olympus, Japan). The setup contains two pulsed diode lasers (LDH405 and LDH470, driver unit 2-channel Sepia II, PicoQuant, Germany) and cw gas laser (HeNe 543 nm, Uniphase, Manteca, CA). Diode lasers offer

The fast polarization modulation based dual-focus fluorescence correlation spectroscopy.

We introduce two new alternative experimental realizations of dual focus fluorescence correlation spectroscopy (2fFCS), a method which allows for obta...
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